Extraction and purification of -galactosidase from

Escherichia coli.
Practical conducted previously aimed to cultivate a strain of E. coli (ATCC 15224) in
bioreactor. This strain has been chosen because it constitutively expresses a protein of interest:
-galactosidase. The purpose of this practical session will be the extraction of galactosidase from the harvested cells.(cellule recoltate)

1. Cell membrane
content(continut)

lysis(liza)

and

expulsion

of

the

intracellular

This step allows extracting the intracellular content of the cells in a buffer suitable for
extraction and separation techniques, such as chromatographic techniques and to maintain the
activity of the enzyme.
600 ml of bacterial culture are centrifuged for 15 minutes at 8000 rpm at room temperature.
Cell pellets are resuspended in sodium acetate/acetic acid 20mM pH4.5 buffer. Final volume
is of 75 ml.
These 75 ml of cells suspended in buffer are sonicated for 10 minutes at a frequency of 0.7
cycles per second at 100% amplitude and at 4 C (in ice). The cell lysate is centrifuged 60
minutes at 10000 rpm at 4C. The supernatant is adjusted to pH 7.5 with 0.5M NaOH and the
volume is noted. A sample (called LF1) of 1 ml is stored at 4 C until assays of protein and
enzyme activity.

2. Precipitation of -galactosidase
An amount of pre-calculated ammonium sulfate salt is added to the intracellular medium
solution with stirring. The ammonium sulfate addition should be done very slowly. Then,
complete the volume to 100 mL with buffer in order to be at 55% of saturation. The solution
is equilibrated at this saturation during 15 minutes at room temperature, and then centrifuged
for 15 minutes at 8000 rpm at room temperature. The pellet is suspended in a minimum
volume of buffer (15 ml) needed to obtain a clear solution. The volume of re-suspended
protein is noted LF2 and a sample of 1 mL is performed and stored at 4C. The same
operation is performed with the precipitation supernatant (noted LF2').
The calculation of the amount of ammonium sulfate is given by the equation:
q = 51.7 (S2-S1) / 1- 0.27S2
with:
- q (g): Quantity of (NH4)2SO4 necessary to obtain the target saturation in 100 mL.
- S1: initial saturation with ammonium sulfate.
- S2: final saturation with ammonium sulfate.

3. Desalting of the product by dialysis

The solution of protein obtained after precipitation is highly concentrated in ammonium
sulfate. This dialysis step aims to remove these salts.
Firstly, a dialysis hose with a length of about 30cm is soaked in distilled water for 15 minutes
before being filled. The content of the hose is then dialyzed against 2,000 ml of buffer
overnight stirring at 4C (in a cold room if possible). The dialysate is then centrifuged at 8000
rpm for 15 minutes at room temperature to remove potential insoluble protein that can be
formed during dialysis. Volume of centrifuged solution is noted (LF3) and a sample of 1 mL
is collected and stored at 4C for assays of proteins and enzyme activity.

4. Expression of results
Monitoring the extraction protocol

Sampl
e
name

Dilution

tota
l
Vol.
(ml)

[protei
n]
(mg/ml
)

Total
Quantity
of
Protein
(mg)

Enzym
e
Activit
y
(U/ml)

Total
Activit
y (U)

Specif
c
Activit
y

Purifca
tion
factor

(U/mg)

LF1
LF2
LF2'
LF3

5. Assays
5.1. Protein Determination by the method of Bradford et al. with Coomassie Blue
Principle
The method is based on the batochrome sliding of absorbance from 465 to 595 nm when
Coomassie Bleu dye binds non-covalently to proteins in acid solution.
Reagents
Bradford reagent (Coomassie Blue). Protein standard solution (BSA Bovine Serum Albumin)
at 2 mg/mL used for the preparation of the BSA standard curve.

Protocol
Perform a standard range of 12 points ranging from 0 to 2000 g / mL, consisting of 8 points
between 0 and 1000 g / mL and 4 points between 1000 and 2000 g / mL (range to achieve 3
times):
Tube n

10

11

12

BSA (L)
H2O
Quantity
(g/ml)
To be
calculated

Transferring 0.1 mL of each tube in a test tube containing 5 mL of

Bradford reagent (Blue Coomassie)
Read the absorbance at 595 nm
Absorbance
series 1 at
595 nm
Absorbance
series 2 at
595 nm
Absorbance
series 3 at
595 nm
Average of
Absorbance

Rince with ethanol

BSA standard curve is plotted and the protein concentration of the samples is determined from
the standard curve.
LF1, LF2, LF2 and LF3 are diluted (if necessary!) in the buffer in test tubes. Then 0.1 mL of
each sample are transferred to 5 ml of Bradford reagent. Absorbance is read at 595 nm against
a blank.
5.2. Assay of enzyme activity
Principle

The -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23) from E. coli

physiologically catalyzes the hydrolysis of lactose to glucose and galactose. However, this
enzyme has a sufficiently broad substrate specificity for hydrolyzing synthetic -Dgalactosides such as o-nitrotrophnyl -D-galactoside (ONPG) to -D-galactose, and onitrophenol (ONP), including the advantage is to absorb at 400 nm.
Reagents
- Buffer acetate sodium/acetic acid 20mM pH4.5
- Solution Na2CO3 1M
- Initial solution of ONP (ONP M = 139.11 g / mol) to 139 mg/l
- ONPG (ONPG M = 301.3 g/mol)
- Prepare a solution of 10 mM ONPG in buffer.
Protocol
An ONP standard concentration range is prepared in triplicate from the stock solution.
Sample

Initial solution (l)

500

100

150

200

500

Buffer (l)

Na2CO3 1M (L)

200

150

100

100

100

100

100

100

Concentration (mg/L)
Absorbance series 1 at 400
nm
Absorbance series 2 at 400
nm
Absorbance series 3 at 400
nm
Average Absorbance

The ONP standard curve is plotted on graph paper.

Assay of -galactosidase in LF samples

The enzyme activity is tested in the samples (LF1, LF2, LF2, LF3) (tests carried out three
times per sample). In a test tube 0.4 ml of 10 mM ONPG were added to 0.5 ml of distilled
water and 2 ml of acetate sodium/acetic acid buffer. Then 0.1mL of enzyme sample (LF1,
LF2, LF2 and LF3) is added and the reaction is stopped after exactly 10 minutes with 1 ml of
1M Na2CO3.

6. Definition of enzyme activity, specific activity and purification factor

The unit of enzyme activity represents the amount of enzyme in 1 ml that is necessary to
catalyze the hydrolysis of 1 mol of ONPG in 1 minute under the conditions of the test (at
room temperature and pH4.5 in buffer).
The specific activity of a fraction is the ratio of the amount of -galactosidase (total enzymatic
activity) on the total amount of proteins in the sample.
The purification factor from one step to the other is logically the ratio of the specific activity
of the fractions across the step.